Individual DNA Molecules Research Articles

Counting DNA molecules on a microchannel surface for quantitative analysis

Microscopic visualization of DNA molecules is a simple, intuitive, and powerful method. Nonetheless, DNA-molecule quantification methods that employ microscopic visualization have not been reported so far. In this study, a new quantitative approach is presented that enables the counting of individual DNA molecules that have been rendered visible by fluorescence microscopy. Toward this, a microfluidic device was employed that directed DNA molecules into microchannels and deposited the molecules onto a positively charged surface. This microfluidic device had a vertically tapered channel inlet structure that prevented the accumulation of excess DNA molecules in the channel inlet while creating a tapering flow, thereby ensuring the even distribution of the DNA molecules in the microchannels. The channel heights and the density of positive charges on the surface were optimized for analysis. The linearity of this method with respect to the determination of the concentration of DNA in solutions was subsequently determined. The limit of detection was 0.48 fg/μL, which corresponds to 64 molecules of 7.25 kbp DNA in 1 μL of sample. This quantitative approach was finally used to count two types of plasmids co-transformed in an E. coli cell; a measurement that is typically considered challenging with gel electrophoresis.

Proton induced DNA double strand breaks at the Bragg peak: Evidence of enhanced LET effect.

PurposeTo investigate DNA double strand breaks (DSBs) induced by therapeutic proton beams in plateau and Bragg peak to demonstrate DSB induction due to the higher LET in the Bragg peak.Materials and MethodspUC19 plasmid DNA samples were irradiated to doses of 1000 and 3000 Gy on a Mevion S250i proton system with a monoenergetic, 110 MeV, proton beam at depths of 2 and 9.4 cm, corresponding to a position on the plateau and distal Bragg peak of the beam, respectively. The irradiated DNA samples were imaged by atomic force microscopy for visualization of individual DNA molecules, either broken or intact, and quantification of the DNA fragment length distributions for each of the irradiated samples. Percentage of the broken DNA and average number of DSBs per DNA molecule were obtained.ResultsCompared to irradiation effects in the plateau region, DNA irradiated at the Bragg peak sustained more breakage at the same dose, yielding more short DNA fragments and higher numbers of DSB per DNA molecule.ConclusionThe higher LET of proton beams at the Bragg peak results in more densely distributed DNA DSBs, which supports an underlying mechanism for the increased cell killing by protons at the Bragg peak.

Biocompatible surface functionalization architecture for a diamond quantum sensor

Quantum metrology enables some of the most precise measurements. In the life sciences, diamond-based quantum sensing has led to a new class of biophysical sensors and diagnostic devices that are being investigated as a platform for cancer screening and ultrasensitive immunoassays. However, a broader application in the life sciences based on nanoscale NMR spectroscopy has been hampered by the need to interface highly sensitive quantum bit (qubit) sensors with their biological targets. Here, we demonstrate an approach that combines quantum engineering with single-molecule biophysics to immobilize individual proteins and DNA molecules on the surface of a bulk diamond crystal that hosts coherent nitrogen vacancy qubit sensors. Our thin (sub-5 nm) functionalization architecture provides precise control over the biomolecule adsorption density and results in near-surface qubit coherence approaching 100 μs. The developed architecture remains chemically stable under physiological conditions for over 5 d, making our technique compatible with most biophysical and biomedical applications.

High molecular weight DNA extraction strategies for long-read sequencing of complex metagenomes.

By offering extremely long contiguous characterization of individual DNA molecules, rapidly emerging long‐read sequencing strategies offer comprehensive insights into the organization of genetic information in genomes and metagenomes. However, successful long‐read sequencing experiments demand high concentrations of highly purified DNA of high molecular weight (HMW), which limits the utility of established DNA extraction kits designed for short‐read sequencing. The challenges associated with input DNA quality intensify further when working with complex environmental samples of low microbial biomass, which requires new protocols that are tailored to study metagenomes with long‐read sequencing. Here, we use human tongue scrapings to benchmark six HMW DNA extraction strategies that are based on commercially available kits, phenol–chloroform (PC) extraction and agarose encasement followed by agarase digestion. A typical end goal of HMW DNA extractions is to obtain the longest possible reads during sequencing, which is often achieved by PC extractions, as demonstrated in sequencing of cultured cells. Yet our analyses that consider overall read‐size distribution, assembly performance and the number of circularized elements found in sequencing results suggest that column‐based kits with enzyme supplementation, rather than PC methods, may be more appropriate for long‐read sequencing of metagenomes.

Real-time imaging and dynamics of single-particle assemblies formed by the AIM2 inflammasome sensor on individual DNA molecules

Cells have developed fundamental mechanisms to sense and react upon foreign substances that indicate the presence of danger. For instance, pathogenic DNA molecules are detected by DNA-sensing proteins that alert the cell, thus enabling preparation for defense. Inflammasomes are large intracellular protein complexes that trigger the inflammatory response as a result of danger signals. Inflammasomes assemble by self-association and oligomerization of multiple copies of three proteins: sensors that respond to danger signals, procaspase-1, which activates cytokines to amplify the inflammatory response, and the adaptor ASC that holds the macromolecular assembly by tethering sensor and procaspase-1 molecules.

Tailored Nucleic Acid Architectures at Gold Surfaces for Controlled Therapeutic Release

Nucleic acids are versatile materials capable of forming smart nanocarriers with highly controllable therapeutic delivery. DNA-gated release is a mechanism by which DNA oligonucleotides physically block the release of encapsulated drugs from porous nanoparticles. We extend this mechanism to be used with drugs bound to the surface of DNA-capped gold nanoparticles (AuNPs). We investigated DNA monolayers of different thicknesses and hybridization states to determine how DNA surface architecture can affect the release of a template drug bound to the gold surface. DNA layers are investigated on the planar gold surface via quartz crystal microbalance with dissipation and on AuNPs via dynamic light scattering. The resultant layer architectures were studied for their effect on the release rate of drugs. We observed that varying DNA architectures on AuNPs result in different release rates of the drug. The rate of drug release can be slowed using either folded or randomly coiled DNA sequences, which act as a physical barrier to diffusion. DNA monolayers with upright orientation release drugs more quickly. When the longer single-stranded DNA is used, the drug release is slowed even further. However, even upright DNA layers provide a barrier to drug diffusion at longer sequence lengths. We hypothesize that it is the architecture of the DNA layer, influenced by the folded or upright orientation of individual DNA molecules, that affects the free diffusion of the drug away from the AuNP surface. This mechanism may improve the biological availability of many surface-bound drugs on solid, DNA-capped nanoparticles.

Prospective molecular methods for sequencing microorganisms in the system of assessment and control of food safety

To solve one of the most important tasks of food hygiene - ensuring foodstuff biosafety, the leading place is given to the use of nucleic acid sequencing methods that allow to identify and describe the nucleotide sequences of genomes both in individual microorganisms and metagenomic characteristics of microbial communities in environmental objects, in humans and animals. The purpose of the review was to analyze the main areas of sequencing application in food microbiology, biotechnology and epidemiology. Material and methods. The collection and analysis of scientific and informational materials published in domestic and foreign publications from Scopus, Web of Science, PubMed, RSCI databases, official reports of the World Health Organization (WHO), the European Food Safety Agency (EFSA) and other published sources has been carried out. Results. The review presents generalized characteristics of the most well-known sequencing formats based on different principles of reading and processing genetic information: capillary sequencing by Sanger, pyrosequencing, sequencing by ligation, sequencing by DNA synthesis, semiconductor high-performance sequencing, sequencing of individual DNA molecules. It is shown that multilocus analysis (MLST, MLVA) increases the specificity of genotyping and intraspecific identification of food-borne pathogens. It is used to assess the pathogenic potential and study the mechanisms of evolutionary variability of bacterial pathogens, leading to polymorphism of clonal lines and the appearance of strain-specific differences, to monitor the different genotypes of pathogens in production and other objects of the food chain. Nanopore sequencing is promising for studying bacterial plasmids and localization of resistance genes of MDR bacteria. Genome-wide analysis (WGS) makes it possible to identify detailed characteristics of pathogens of infectious diseases, including uncultivated and previously unknown microorganisms, as well as to study the features of the organization of individual pro- and eukaryotic genomes, gene regulation systems, pathogenicity factors and protein expression. The role of WGS methods for determining the degree of genetic kinship of infectious agents isolated from various sources in the investigation of outbreaks and sporadic food poisoning and infections is shown. The possibilities of metagenomic sequencing in the study of species composition, genetic diversity and metabolic potential of microbial communities are demonstrated. The main advantage of metagenomics is the possibility of direct sequencing of the genomes of microorganisms living in the human body, animals, water, soil and other objects. Metagenomic analysis plays a leading role in the detection of unexplored taxa, uncultivated and difficult-to-cultivate forms of a wide range of microorganisms. The most important direction of metagenomics is the study of the human or animal microbiome, the analysis of the relationship between the intestinal microbiota and metabolism, as well as the formation of the nutritional status of a person. Theoretical and practical aspects of the implementation of high-performance sequencing methods in the system of food biosafety assessment and control are reviewed: safety assessment of genetically modified strains of food producers, development of fundamentally new algorithms for investigating outbreaks of infectious diseases, creation of network systems for early diagnosis and notification of food infections of bacterial and viral etiology. Conclusion. Sequencing should become a standard methodology in the field of food safety for the identification and characterization of food pathogens, including those with resistance to antibiotics and adverse technological effects. The introduction of WGS into the food safety assessment and control system will make it possible to assess real food contamination with new and newly returning pathogens, create electronic databases and scientifically substantiate the most adequate measures for the prevention of food infections and ensuring the health of the population.

Combining dense and sparse labeling in optical DNA mapping.

Optical DNA mapping (ODM) is based on fluorescent labeling, stretching and imaging of single DNA molecules to obtain sequence-specific fluorescence profiles, DNA barcodes. These barcodes can be mapped to theoretical counterparts obtained from DNA reference sequences, which in turn allow for DNA identification in complex samples and for detecting structural changes in individual DNA molecules. There are several types of DNA labeling schemes for ODM and for each labeling type one or several types of match scoring methods are used. By combining the information from multiple labeling schemes one can potentially improve mapping confidence; however, combining match scores from different labeling assays has not been implemented yet. In this study, we introduce two theoretical methods for dealing with analysis of DNA molecules with multiple label types. In our first method, we convert the alignment scores, given as output from the different assays, into p-values using carefully crafted null models. We then combine the p-values for different label types using standard methods to obtain a combined match score and an associated combined p-value. In the second method, we use a block bootstrap approach to check for the uniqueness of a match to a database for all barcodes matching with a combined p-value below a predefined threshold. For obtaining experimental dual-labeled DNA barcodes, we introduce a novel assay where we cut plasmid DNA molecules from bacteria with restriction enzymes and the cut sites serve as sequence-specific markers, which together with barcodes obtained using the established competitive binding labeling method, form a dual-labeled barcode. All experimental data in this study originates from this assay, but we point out that our theoretical framework can be used to combine data from all kinds of available optical DNA mapping assays. We test our multiple labeling frameworks on barcodes from two different plasmids and synthetically generated barcodes (combined competitive-binding- and nick-labeling). It is demonstrated that by simultaneously using the information from all label types, we can substantially increase the significance when we match experimental barcodes to a database consisting of theoretical barcodes for all sequenced plasmids.

DNA bridges: A novel platform for single-molecule sequencing and other DNA-protein interaction applications.

DNA molecular combing is a technique that stretches thousands of long individual DNA molecules (up to 10 Mbp) into a parallel configuration on surface. It has previously been proposed to sequence these molecules by synthesis. However, this approach poses two critical challenges: 1-Combed DNA molecules are overstretched and therefore a nonoptimal substrate for polymerase extension. 2-The combing surface sterically impedes full enzymatic access to the DNA backbone. Here, we introduce a novel approach that attaches thousands of molecules to a removable surface, with a tunable stretching factor. Next, we dissolve portions of the surface, leaving the DNA molecules suspended as ‘bridges’. We demonstrate that the suspended molecules are enzymatically accessible, and we have used an enzyme to incorporate labeled nucleotides, as predicted by the specific molecular sequence. Our results suggest that this novel platform is a promising candidate to achieve high-throughput sequencing of Mbp-long molecules, which could have additional genomic applications, such as the study of other protein-DNA interactions.

Phase 2 Study of Acalabrutinib Window Prior to Frontline Therapy in Untreated Aggressive B-Cell Lymphoma: Preliminary Results and Correlatives of Response to Acalabrutinib

Phase 2 Study of Acalabrutinib Window Prior to Frontline Therapy in Untreated Aggressive B-Cell Lymphoma: Preliminary Results and Correlatives of Response to Acalabrutinib

A Minimalistic Coumarin Turn-On Probe for Selective Recognition of Parallel G-Quadruplex DNA Structures.

G-quadruplex (G4) DNA structures are widespread in the human genome and are implicated in biologically important processes such as telomere maintenance, gene regulation, and DNA replication. Guanine-rich sequences with potential to form G4 structures are prevalent in the promoter regions of oncogenes, and G4 sites are now considered as attractive targets for anticancer therapies. However, there are very few reports of small “druglike” optical G4 reporters that are easily accessible through one-step synthesis and that are capable of discriminating between different G4 topologies. Here, we present a small water-soluble light-up fluorescent probe that features a minimalistic amidinocoumarin-based molecular scaffold that selectively targets parallel G4 structures over antiparallel and non-G4 structures. We showed that this biocompatible ligand is able to selectively stabilize the G4 template resulting in slower DNA synthesis. By tracking individual DNA molecules, we demonstrated that the G4-stabilizing ligand perturbs DNA replication in cancer cells, resulting in decreased cell viability. Moreover, the fast-cellular entry of the probe enabled detection of nucleolar G4 structures in living cells. Finally, insights gained from the structure–activity relationships of the probe suggest the basis for the recognition of parallel G4s, opening up new avenues for the design of new biocompatible G4-specific small molecules for G4-driven theranostic applications.

Long reads capture simultaneous enhancer-promoter methylation status for cell-type deconvolution.

MotivationWhile promoter methylation is associated with reinforcing fundamental tissue identities, the methylation status of distant enhancers was shown by genome-wide association studies to be a powerful determinant of cell-state and cancer. With recent availability of long reads that report on the methylation status of enhancer–promoter pairs on the same molecule, we hypothesized that probing these pairs on the single-molecule level may serve the basis for detection of rare cancerous transformations in a given cell population. We explore various analysis approaches for deconvolving cell-type mixtures based on their genome-wide enhancer–promoter methylation profiles.ResultsTo evaluate our hypothesis we examine long-read optical methylome data for the GM12878 cell line and myoblast cell lines from two donors. We identified over 100 000 enhancer–promoter pairs that co-exist on at least 30 individual DNA molecules. We developed a detailed methodology for mixture deconvolution and applied it to estimate the proportional cell compositions in synthetic mixtures. Analysis of promoter methylation, as well as enhancer–promoter pairwise methylation, resulted in very accurate estimates. In addition, we show that pairwise methylation analysis can be generalized from deconvolving different cell types to subtle scenarios where one wishes to resolve different cell populations of the same cell-type.Availability and implementationThe code used in this work to analyze single-molecule Bionano Genomics optical maps is available via the GitHub repository https://github.com/ebensteinLab/Single_molecule_methylation_in_EP.

Atomic force microscopy-A tool for structural and translational DNA research.

Atomic force microscopy (AFM) is a powerful imaging technique that allows for structural characterization of single biomolecules with nanoscale resolution. AFM has a unique capability to image biological molecules in their native states under physiological conditions without the need for labeling or averaging. DNA has been extensively imaged with AFM from early single-molecule studies of conformational diversity in plasmids, to recent examinations of intramolecular variation between groove depths within an individual DNA molecule. The ability to image dynamic biological interactions in situ has also allowed for the interaction of various proteins and therapeutic ligands with DNA to be evaluated—providing insights into structural assembly, flexibility, and movement. This review provides an overview of how innovation and optimization in AFM imaging have advanced our understanding of DNA structure, mechanics, and interactions. These include studies of the secondary and tertiary structure of DNA, including how these are affected by its interactions with proteins. The broader role of AFM as a tool in translational cancer research is also explored through its use in imaging DNA with key chemotherapeutic ligands, including those currently employed in clinical practice.

Covalent Positioning of Single DNA Molecules for Nanopatterning.

Bottom-up micropatterning or nanopatterning can be viewed as the localization of target molecules to the desired area of a surface. A majority of these processes rely on the physical adsorption of ink-like molecules to the paper-like surface, resulting in unstable immobilization of the target molecules owing to their noncovalent linkage to the surface. Herein, successive single nick-sealing facilitated the covalent immobilization of individual DNA molecules at defined positions on a dendron-coated silicon surface using atomic force microscopy. The covalently-patterned ssDNA was visualized when the streptavidin-coated gold nanoparticles bound to the biotinylated DNA. The successive covalent positioning of the target DNA under ambient conditions may facilitate the bottom-up construction of DNA-based durable nanostructures, nanorobots, or memory system.

Single-molecule optical genome mapping in nanochannels: multidisciplinarity at the nanoscale.

The human genome contains multiple layers of information that extend beyond the genetic sequence. In fact, identical genetics do not necessarily yield identical phenotypes as evident for the case of two different cell types in the human body. The great variation in structure and function displayed by cells with identical genetic background is attributed to additional genomic information content. This includes large-scale genetic aberrations, as well as diverse epigenetic patterns that are crucial for regulating specific cell functions. These genetic and epigenetic patterns operate in concert in order to maintain specific cellular functions in health and disease. Single-molecule optical genome mapping is a high-throughput genome analysis method that is based on imaging long chromosomal fragments stretched in nanochannel arrays. The access to long DNA molecules coupled with fluorescent tagging of various genomic information presents a unique opportunity to study genetic and epigenetic patterns in the genome at a single-molecule level over large genomic distances. Optical mapping entwines synergistically chemical, physical, and computational advancements, to uncover invaluable biological insights, inaccessible by sequencing technologies. Here we describe the method’s basic principles of operation, and review the various available mechanisms to fluorescently tag genomic information. We present some of the recent biological and clinical impact enabled by optical mapping and present recent approaches for increasing the method’s resolution and accuracy. Finally, we discuss how multiple layers of genomic information may be mapped simultaneously on the same DNA molecule, thus paving the way for characterizing multiple genomic observables on individual DNA molecules.

Single-molecule optical mapping of the distribution of DNA phosphorothioate epigenetics

DNA phosphorothioate (PT) modifications, with the nonbridging phosphate oxygen replaced by sulfur, governed by DndABCDE or SspABCD, are widely distributed in prokaryotes and have a highly unusual feature of occupying only a small portion of available consensus sequences in a genome. Despite the presence of plentiful non-PT-protected consensuses, DNA PT modification is still employed as a recognition tag by the restriction cognate, for example, DndFGH or SspE, to discriminate and destroy PT-lacking foreign DNA. This raises a fundamental question about how PT modifications are distributed along DNA molecules to keep the restriction components in check. Here, we present two single-molecule strategies that take advantage of the nucleophilicity of PT in combination with fluorescent markers for optical mapping of both single- and double-stranded PT modifications across individual DNA molecules. Surprisingly, PT profiles vary markedly from molecule to molecule, with different PT locations and spacing distances between PT pairs, even in the presence of DndFGH or SspE. The results revealed unprecedented PT modification features previously obscured by ensemble averaging, providing novel insights into the riddles regarding unusual target selection by PT modification and restriction components.

Magnetic Nanoparticles as a Tool for Remote DNA Manipulations at a Single-Molecule Level.

Remote control of cells and single molecules by magnetic nanoparticles in nonheating external magnetic fields is a perspective approach for many applications such as cancer treatment and enzyme activity regulation. However, the possibility and mechanisms of direct effects of small individual magnetic nanoparticles on such processes in magneto-mechanical experiments still remain unclear. In this work, we have shown remote-controlled mechanical dissociation of short DNA duplexes (18-60 bp) under the influence of nonheating low-frequency alternating magnetic fields using individual 11 nm magnetic nanoparticles. The developed technique allows (1) simultaneous manipulation of millions of individual DNA molecules and (2) evaluation of energies of intermolecular interactions in short DNA duplexes or in other molecules. Finally, we have shown that DNA duplexes dissociation is mediated by mechanical stress and produced by the movement of magnetic nanoparticles in magnetic fields, but not by local overheating. The presented technique opens a new avenue for high-precision manipulation of DNA and generation of biosensors for quantification of energies of intermolecular interaction.

DNA length tunes the fluidity of DNA-based condensates

Living organisms typically store their genomic DNA in a condensed form. Mechanistically, DNA condensation can be driven by macromolecular crowding, multivalent cations, or positively charged proteins. At low DNA concentration, condensation triggers the conformational change of individual DNA molecules into a compacted state, with distinct morphologies. Above a critical DNA concentration, condensation goes along with phase separation into a DNA-dilute and a DNA-dense phase. The latter DNA-dense phase can have different material properties and has been reported to be rather liquid-like or solid-like depending on the characteristics of the DNA and the solvent composition. Here, we systematically assess the influence of DNA length on the properties of the resulting condensates. We show that short DNA molecules with sizes below 1 kb can form dynamic liquid-like assemblies when condensation is triggered by polyethylene glycol and magnesium ions, binding of linker histone H1, or nucleosome reconstitution in combination with linker histone H1. With increasing DNA length, molecules preferentially condense into less dynamic more solid-like assemblies, with phage λ-DNA with 48.5 kb forming mostly solid-like assemblies under the conditions assessed here. The transition from liquid-like to solid-like condensates appears to be gradual, with DNA molecules of roughly 1–10 kb forming condensates with intermediate properties. Titration experiments with linker histone H1 suggest that the fluidity of condensates depends on the net number of attractive interactions established by each DNA molecule. We conclude that DNA molecules that are much shorter than a typical human gene are able to undergo liquid-liquid phase separation, whereas longer DNA molecules phase separate by default into rather solid-like condensates. We speculate that the local distribution of condensing factors can modulate the effective length of chromosomal domains in the cell. We anticipate that the link between DNA length and fluidity established here will improve our understanding of biomolecular condensates involving DNA.

Intra-host changes in Kaposi sarcoma-associated herpesvirus genomes in Ugandan adults with Kaposi sarcoma.

Intra-host tumor virus variants may influence the pathogenesis and treatment responses of some virally-associated cancers. However, the intra-host variability of Kaposi sarcoma-associated herpesvirus (KSHV), the etiologic agent of Kaposi sarcoma (KS), has to date been explored with sequencing technologies that possibly introduce more errors than that which occurs in the viral population, and these studies have only studied variable regions. Here, full-length KSHV genomes in tumors and/or oral swabs from 9 Ugandan adults with HIV-associated KS were characterized. Furthermore, we used deep, short-read sequencing using duplex unique molecular identifiers (dUMI)-random double-stranded oligonucleotides that barcode individual DNA molecules before library amplification. This allowed suppression of PCR and sequencing errors to ~10-9/base as well as afforded accurate determination of KSHV genome numbers sequenced in each sample. KSHV genomes were assembled de novo, and rearrangements observed were confirmed by PCR and Sanger sequencing. 131-kb KSHV genome sequences, excluding major repeat regions, were successfully obtained from 23 clinical specimens, averaging 2.3x104 reads/base. Strikingly, KSHV genomes were virtually identical within individuals at the point mutational level. The intra-host heterogeneity that was observed was confined to tumor-associated KSHV mutations and genome rearrangements, all impacting protein-coding sequences. Although it is unclear whether these changes were important to tumorigenesis or occurred as a result of genomic instability in tumors, similar changes were observed across individuals. These included inactivation of the K8.1 gene in tumors of 3 individuals and retention of a region around the first major internal repeat (IR1) in all instances of genomic deletions and rearrangements. Notably, the same breakpoint junctions were found in distinct tumors within single individuals, suggesting metastatic spread of rearranged KSHV genomes. These findings define KSHV intra-host heterogeneity in vivo with greater precision than has been possible in the past and suggest the possibility that aberrant KSHV genomes may contribute to aspects of KS tumorigenesis. Furthermore, study of KSHV with use of dUMI provides a proof of concept for utilizing this technique for detailed study of other virus populations in vivo.

FLT3 Internal Tandem Duplication Quantitation and Length Analysis By Digital PCR Paired with High-Speed AFM

Introduction DNA length polymorphisms are difficult to diagnose due to their repetitive or variable elements. Within the FLT3 gene, internal tandem duplications (ITDs) range from <30 bp to >200 bp in length and are associated with poor prognosis in acute myeloid leukemia (AML). To address these challenges, we developed digital polymerase chain reaction (dPCR) followed by high-speed atomic force microscopy (HSAFM) as a high-throughput, single-molecule approach for quantifying and sizing length polymorphisms associated with FLT3-ITDs. Methods In the first step of our approach, dPCR (Fig. 1A), a mixed sample is diluted and partitioned into micro-reactions that are individually amplified, mitigating amplification bias and producing homogeneous solutions of either wild-type (WT) or FLT3-ITD variant amplicons. We then used HSAFM to directly image individual amplicons and determine their lengths with nanoscale resolution (Fig. 1B). Length distributions were analyzed by Bayesian inference to identify their most likely character-WT or variant-by determining whether or not the 95% most credible range of mean lengths for each micro-reaction fell within a region of practical equivalence (ROPE) to WT length (Fig. 1C). Variant allele frequency (VAF) was then calculated as the proportion of variant-identified micro-reactions (Fig. 1D). We tested (a) synthetic and cell line DNA ranging from 30 bp to 217 bp, with VAF titrated down to 5%, and (b) clinical FLT3-ITD positive and negative samples, and compared dPCR-HSAFM results to those of a widespread clinical assay (Invivoscribe Leukostrat CDx FLT3 Mutation Assay). Results dPCR-HSAFM successfully returned the VAFs of all spiked-in FLT3-ITD samples across the admixture range of 5%-100%, and WT-only controls yielded VAFs of 0% (Fig. 1C,D). The four tested clinical samples determined as FLT3-ITD positive by the standard assay returned VAFs of 49%, 16%, 26%, and 61% by dPCR-HSAFM, while the two FLT3-ITD negative clinical samples returned VAFs of 6% and <1% (Fig. 1C). Mean variant length of control and clinical samples measured by dPCR-HSAFM agreed with the known lengths as reported by the Leukostrat assay or given by cell line documentation (Fig. 1C, horizontal lines). Conclusions By demonstrating accurate quantification and sizing of FLT3-ITD variants of clinically relevant size and VAF, dPCR-HSAFM supersedes the standard clinical assay, which does not report VAF. Since FLT3-ITD length and VAF are linked to AML outcome and are important for minimal residual disease following therapy, a test that provides both metrics is desirable. Furthermore, the speed, low cost, and flexibility of dPCR-HSAFM to detect long, repetitive insertions make it an attractive alternative to sequencing in the clinical diagnosis of FLT3-ITDs and other length polymorphisms. Figure 1. dCR-HSAFM technique and results. (A) Digital PCR of a mixed sample yields a homogeneous population of WT (blue) or variant FLT3-ITD (red) amplicons in each micro-reaction. (B) HSAFM images of DNA are traced with custom software to yield lengths of individual DNA molecules. (C) Length is used to classify each dPCR reaction as WT or variant, where each vertical line gives the 95% most credible range of mean length for a dPCR reaction and italicized numbers give the number of dPCR reactions for each sample. The four leftmost clinical samples were identified as FLT3-ITD positive by a standard clinical assay, while the two rightmost clinical samples were FLT3-ITD negative. The assay does not report VAF. (D) Spiked-in VAF v. detected VAF is shown for FLT3-ITD samples with 30 bp and 126 bp insertions, with spiked-in VAF ranging from 5%-100%. Vertical bars give the VAFs for ROPEs ± 10% of the initial ROPE width, which conveys the sensitivity of each VAF to the ROPE width. Figure 1 Disclosures No relevant conflicts of interest to declare.